dsrna viruses
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2022 ◽  
Author(s):  
Mark D Lee ◽  
Jack W Creagh ◽  
Lance R Fredericks ◽  
Angela M Crabtree ◽  
Jagsish Suresh Patel ◽  
...  

Mycoviruses are widely distributed across fungi, including yeasts of the Saccharomycotina subphylum. It was recently discovered that the yeast species Pichia membranifaciens contained double stranded RNAs (dsRNAs) that were predicted to be of viral origin. The fully sequenced dsRNA is 4,578 bp in length, with RNA secondary structures similar to the packaging, replication, and frameshift signals of totiviruses of the family Totiviridae. This novel virus has been named Pichia membranifaciens virus L-A (PmV-L-A) and is related to other totiviruses previously described within the Saccharomycotina yeasts. PmV-L-A is part of a monophyletic subgroup within the I-A totiviruses, implying a common ancestry between mycoviruses isolated from the Pichiaceae and Saccharomycetaceae yeasts. Energy minimized AlphaFold2 molecular models of the PmV-L-A Gag protein revealed structural conservation with the previously solved structure of the Saccharomyces cerevisiae virus L-A (ScV-L-A) Gag protein. The predicted tertiary structure of the PmV-L-A Pol and its homologs provide details of the potential mechanism of totivirus RNA-dependent RNA polymerases (RdRps) because of structural similarities to the RdRps of mammalian dsRNA viruses. Insights into the structure, function, and evolution of totiviruses gained from yeasts is important because of their parallels with mammalian viruses and the emerging role of totiviruses in animal disease.


2021 ◽  
Author(s):  
María C. Gimenez ◽  
Yesica R. Frontini-Lopez ◽  
Cristian A. Pocognoni ◽  
Julieta S. Roldán ◽  
Clara García Samartino ◽  
...  

Birnaviruses are members of the Birnaviridae family, responsible for major economic losses to poultry and aquaculture. The family is composed of non-enveloped viruses with a segmented double-stranded RNA (dsRNA) genome. Infectious bursal disease virus (IBDV), the prototypic family member, is the etiological agent of Gumboro disease, a highly contagious immunosuppressive disease in the poultry industry worldwide. We previously demonstrated that IBDV hijacks the endocytic pathway for establishing the viral replication complexes on endosomes associated with the G olgi c omplex (GC). In this work, we report that IBDV reorganizes the GC to localize the endosome-associated replication complexes without affecting its secretory functionality. Analyzing crucial proteins involved in the secretory pathway, we showed the essential requirement of Rab1b for viral replication. Rab1b comprises a key regulator of GC transport and we demonstrate that transfecting the negative mutant Rab1b N121I or knocking down Rab1b expression by RNA interference significantly reduces the yield of infectious viral progeny. Furthermore, we showed that the Rab1b downstream effector G olgi-specific B FA resistance f actor 1 (GBF1), which activates the small GTPase A DP- r ibosylation f actor 1 (ARF1), is required for IBDV replication since inhibiting its activity by treatment with b re f eldin A (BFA) or G olgi c ide A (GCA) significantly reduces the yield of infectious viral progeny. Finally, we show that ARF1 dominant negative-mutant T31N over-expression hampered the IBDV infection. Taken together, these results demonstrate that IBDV requires the function of the Rab1b-GBF1-ARF1 axis to promote its replication, making a substantial contribution to the field of birnaviruses-host cell interactions. IMPORTANCE Birnaviruses are unconventional members of the dsRNA viruses, being the lack of a transcriptionally active core the main differential feature. This structural trait, among others that resemble the plus single-stranded (+ssRNA) viruses features, suggests that birnaviruses might follow a different replication program from that conducted by prototypical dsRNA members and have argued the hypothesis that birnaviruses could be evolutionary links between +ssRNA and dsRNA viruses. Here, we present original data showing the IBDV-induced GC reorganization and the crosstalk between IBDV and the Rab1b-GBF1-ARF1 mediated intracellular trafficking pathway. The replication of several +ssRNA viruses depends on the cellular protein GBF1, but its role in the replication process is not clear. Thus, our findings make a substantial contribution to the field of birnaviruses-host cells and provide further evidence supporting the proposed evolutionary connection role of birnaviruses, an aspect which we consider especially relevant for researchers working in the virology field.


Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2319
Author(s):  
Tünde Kartali ◽  
Ildikó Nyilasi ◽  
Sándor Kocsubé ◽  
Roland Patai ◽  
Tamás F. Polgár ◽  
...  

We previously screened the total nucleic acid extracts of 123 Mucor strains for the presence of dsRNA molecules without further molecular analyses. Here, we characterized five novel dsRNA genomes isolated from four different Mucor hiemalis strains with next-generation sequencing (NGS), namely Mucor hiemalis virus 1a (MhV1a) from WRL CN(M) 122; Mucor hiemalis virus 1b (MhV1b) from NRRL 3624; Mucor hiemalis virus 2 (MhV2) from NRRL 3616; and Mucor hiemalis virus 3 (MhV3) and Mucor hiemalis virus (MhV4) from NRRL 3617 strains. Genomes contain two open reading frames (ORF), which encode the coat protein (CP) and the RNA dependent RNA polymerase (RdRp), respectively. In MhV1a and MhV1b, it is predicted to be translated as a fusion protein via -1 ribosomal frameshift, while in MhV4 via a rare +1 (or−2) ribosomal frameshift. In MhV2 and MhV3, the presence of specific UAAUG pentanucleotide motif points to the fact for coupled translation termination and reinitialization. MhV1a, MhV2, and MhV3 are part of the clade representing the genus Victorivirus, while MhV4 is seated in Totivirus genus clade. The detected VLPs in Mucor strains were from 33 to 36 nm in diameter. Hybridization analysis revealed that the dsRNA molecules of MhV1a-MhV4 hybridized to the corresponding molecules.


2021 ◽  
Vol 12 ◽  
Author(s):  
Luc Swevers ◽  
Dimitrios Kontogiannatos ◽  
Anna Kolliopoulou ◽  
Feifei Ren ◽  
Min Feng ◽  
...  

While RNAi is often heralded as a promising new strategy for insect pest control, a major obstacle that still remains is the efficient delivery of dsRNA molecules within the cells of the targeted insects. However, it seems overlooked that dsRNA viruses already have developed efficient strategies for transport of dsRNA molecules across tissue barriers and cellular membranes. Besides protecting their dsRNA genomes in a protective shell, dsRNA viruses also display outer capsid layers that incorporate sophisticated mechanisms to disrupt the plasma membrane layer and to translocate core particles (with linear dsRNA genome fragments) within the cytoplasm. Because of the perceived efficiency of the translocation mechanism, it is well worth analyzing in detail the molecular processes that are used to achieve this feat. In this review, the mechanism of cell entry by dsRNA viruses belonging to the Reoviridae family is discussed in detail. Because of the large amount of progress in mammalian versus insect models, the mechanism of infections of reoviruses in mammals (orthoreoviruses, rotaviruses, orbiviruses) will be treated as a point of reference against which infections of reoviruses in insects (orbiviruses in midges, plant viruses in hemipterans, insect-specific cypoviruses in lepidopterans) will be compared. The goal of this discussion is to uncover the basic principles by which dsRNA viruses cross tissue barriers and translocate their cargo to the cellular cytoplasm; such knowledge subsequently can be incorporated into the design of dsRNA virus-based viral-like particles for optimal delivery of RNAi triggers in targeted insect pests.


2021 ◽  
Vol 102 (7) ◽  
Author(s):  
Caitlin A. O'Brien ◽  
Jessica J. Harrison ◽  
Agathe M. G. Colmant ◽  
Renee J. Traves ◽  
Devina Paramitha ◽  
...  

Mosquito-borne flaviviruses are significant contributors to the arboviral disease burdens both in Australia and globally. While routine arbovirus surveillance remains a vital exercise to identify known flaviviruses in mosquito populations, novel or divergent and emerging species can be missed by these traditional methods. The MAVRIC (monoclonal antibodies to viral RNA intermediates in cells) system is an ELISA-based method for broad-spectrum isolation of positive-sense and double-stranded RNA (dsRNA) viruses based on detection of dsRNA in infected cells. While the MAVRIC ELISA has successfully been used to detect known and novel flaviviruses in Australian mosquitoes, we previously reported that dsRNA could not be detected in dengue virus-infected cells using this method. In this study we identified additional flaviviruses which evade detection of dsRNA by the MAVRIC ELISA. Utilising chimeric flaviviruses we demonstrated that this outcome may be dictated by the non-structural proteins and/or untranslated regions of the flaviviral genome. In addition, we report a modified fixation method that enables improved detection of flavivirus dsRNA and inactivation of non-enveloped viruses from mosquito populations using the MAVRIC system. This study demonstrates the utility of anti-dsRNA monoclonal antibodies for identifying viral replication in insect and vertebrate cell systems and highlights a unique characteristic of flavivirus replication.


2021 ◽  
Author(s):  
Xiaoyao Cai ◽  
Fengjuan Tian ◽  
Li Teng ◽  
Hongmei Liu ◽  
Yigang Tong ◽  
...  

Bacteriophages are considered the most abundant entities on earth. However, there are merely seven sequenced double-stranded (ds)RNA phages compared with thousands of dsDNA phages. Interestingly, dsRNA viruses are quite common in fungi and usually have a lifestyle of commensalism or mutualism. Thus, the classical protocol of using double-layer agar plates to characterize phage plaques might be significantly biased in the isolation of dsRNA phages beyond strictly lytic lifestyles. Thus, we applied the protocol of isolating fungal viruses to identify RNA phages in bacteria and successfully isolated a novel dsRNA phage, phiNY, from Microvirgula aerodenitrificans . phiNY has a genome of three dsRNA segments, and its genome sequence has no nucleotide sequence similarity with any other phage. Although phiNY encodes a lytic protein of glycoside hydrolase and phage particles are consistently released during bacterial growth, phiNY replication did not block bacteria growth, nor did it form any plaque on agar plates. More strikingly, the phiNY-infected strain grew faster than the phiNY-negative strain, indicating a mutualistic parasitic lifestyle. Thus, this study not only reveals a new mutualistic parasitic dsRNA phage but also implies that other virus isolation methods would be valuable to identify phages with other lifestyles. Importance Viruses with dsRNA genomes are quite diverse and infect organisms in all three domains of life. Though dsRNA viruses infecting humans, plants and fungi are quite common, dsRNA viruses infecting bacteria, known as bacteriophages, are quite understudied and only seven dsRNA phages have been sequenced so far. One possible explanation for the rare isolation of dsRNA phages might be the protocols of double-layer agar plates assay. Phages beyond strictly lytic lifestyles might not form plaques. Thus, we applied the protocol of isolating fungal viruses to identify RNA phages inside bacteria and successfully isolated a novel dsRNA phage phiNY with a mutualistic parasitic lifestyle. This study implies dsRNA phages beyond strictly lytic lifestyle might be common in nature and deserves more investigations.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Riikka Linnakoski ◽  
Suvi Sutela ◽  
Martin P. A. Coetzee ◽  
Tuan A. Duong ◽  
Igor N. Pavlov ◽  
...  

AbstractSpecies of Armillaria are distributed globally and include some of the most important pathogens of forest and ornamental trees. Some of them form large long-living clones that are considered as one of the largest organisms on earth and are capable of long-range spore-mediated transfer as well as vegetative spread by drought-resistant hyphal cords called rhizomorphs. However, the virus community infecting these species has remained unknown. In this study we used dsRNA screening and high-throughput sequencing to search for possible virus infections in a collection of Armillaria isolates representing three different species: Armillaria mellea from South Africa, A. borealis from Finland and Russia (Siberia) and A. cepistipes from Finland. Our analysis revealed the presence of both negative-sense RNA viruses and positive-sense RNA viruses, while no dsRNA viruses were detected. The viruses included putative new members of virus families Mymonaviridae, Botourmiaviridae and Virgaviridae and members of a recently discovered virus group tentatively named “ambiviruses” with ambisense bicistronic genomic organization. We demonstrated that Armillaria isolates can be cured of viruses by thermal treatment, which enables the examination of virus effects on host growth and phenotype using isogenic virus-infected and virus-free strains.


mBio ◽  
2021 ◽  
Vol 12 (2) ◽  
Author(s):  
Alexander Stevens ◽  
Katherine Muratore ◽  
Yanxiang Cui ◽  
Patricia J. Johnson ◽  
Z. Hong Zhou

ABSTRACT Trichomonas vaginalis, the causative pathogen for the most common nonviral sexually transmitted infection worldwide, is itself frequently infected with one or more of the four types of small double-stranded RNA (dsRNA) Trichomonas vaginalis viruses (TVV1 to 4, genus Trichomonasvirus, family Totiviridae). Each TVV encloses a nonsegmented genome within a single-layered capsid and replicates entirely intracellularly, like many dsRNA viruses, and unlike those in the Reoviridae family. Here, we have determined the structure of TVV2 by cryo-electron microscopy (cryoEM) at 3.6 Å resolution and derived an atomic model of its capsid. TVV2 has an icosahedral, T = 2*, capsid comprised of 60 copies of the icosahedral asymmetric unit (a dimer of the two capsid shell protein [CSP] conformers, CSP-A and CSP-B), typical of icosahedral dsRNA virus capsids. However, unlike the robust CSP-interlocking interactions such as the use of auxiliary “clamping” proteins among Reoviridae, only lateral CSP interactions are observed in TVV2, consistent with an assembly strategy optimized for TVVs’ intracellular-only replication cycles within their protozoan host. The atomic model reveals both a mostly negatively charged capsid interior, which is conducive to movement of the loosely packed genome, and channels at the 5-fold vertices, which we suggest as routes of mRNA release during transcription. Structural comparison of TVV2 to the Saccharomyces cerevisiae L-A virus reveals a conserved helix-rich fold within the CSP and putative guanylyltransferase domain along the capsid exterior, suggesting conserved mRNA maintenance strategies among Totiviridae. This first atomic structure of a TVV provides a framework to guide future biochemical investigations into the interplay between Trichomonas vaginalis and its viruses. IMPORTANCE Trichomonas vaginalis viruses (TVVs) are double-stranded RNA (dsRNA) viruses that cohabitate in Trichomonas vaginalis, the causative pathogen of trichomoniasis, the most common nonviral sexually transmitted disease worldwide. Featuring an unsegmented dsRNA genome encoding a single capsid shell protein (CSP), TVVs contrast with multisegmented dsRNA viruses, such as the diarrhea-causing rotavirus, whose larger genome is split into 10 dsRNA segments encoding 5 unique capsid proteins. To determine how TVVs incorporate the requisite functionalities for viral replication into their limited proteome, we derived the atomic model of TVV2, a first for TVVs. Our results reveal the intersubunit interactions driving CSP association for capsid assembly and the properties that govern organization and maintenance of the viral genome. Structural comparison between TVV2 capsids and those of distantly related dsRNA viruses indicates conserved strategies of nascent RNA release and a putative viral guanylyltransferase domain implicated in the cytoplasmic maintenance of viral messenger and genomic RNA.


Author(s):  
Claudia Buser ◽  
Jukka Jokela ◽  
Oliver Martin

Vector-borne parasites often manipulate hosts to attract uninfected vectors. For example, parasites causing malaria alter host odor to attract mosquitoes. Here we discuss the ecology and evolution of fruit-colonizing yeast in a tripartite symbiosis – the so-called “killer yeast” system. “Killer yeast” consists of Saccharomyces cerevisiae yeast hosting two double stranded RNA viruses (M satellite dsRNAs, L-A dsRNA helper virus). When both dsRNA viruses occur in a yeast cell, the yeast converts to lethal toxin‑producing “killer yeast” phenotype that kills uninfected yeasts. Yeasts on ephemeral fruits attract insect vectors to colonize new habitats. As the viruses have no extracellular stage, they depend on the same insect vectors as yeast for their dispersal. Viruses also benefit from yeast dispersal as this promotes yeast to reproduce sexually, which is how viruses can transmit to uninfected yeast strains. We tested whether insect vectors are more attracted to killer yeasts than to non‑killer yeasts. In our field experiment, we found that killer yeasts were more attractive to Drosophila than non-killer yeasts. This suggests that vectors foraging on yeast are more likely to transmit yeast with a killer phenotype, allowing the viruses to colonize those uninfected yeast strains that engage in sexual reproduction with the killer yeast. Beyond insights into the basic ecology of the killer yeast system, our results suggest that viruses could increase transmission success by manipulating the insect vectors of their host.


PLoS Genetics ◽  
2021 ◽  
Vol 17 (2) ◽  
pp. e1009341 ◽  
Author(s):  
Lance R. Fredericks ◽  
Mark D. Lee ◽  
Angela M. Crabtree ◽  
Josephine M. Boyer ◽  
Emily A. Kizer ◽  
...  

Killer toxins are extracellular antifungal proteins that are produced by a wide variety of fungi, including Saccharomyces yeasts. Although many Saccharomyces killer toxins have been previously identified, their evolutionary origins remain uncertain given that many of these genes have been mobilized by double-stranded RNA (dsRNA) viruses. A survey of yeasts from the Saccharomyces genus has identified a novel killer toxin with a unique spectrum of activity produced by Saccharomyces paradoxus. The expression of this killer toxin is associated with the presence of a dsRNA totivirus and a satellite dsRNA. Genetic sequencing of the satellite dsRNA confirmed that it encodes a killer toxin with homology to the canonical ionophoric K1 toxin from Saccharomyces cerevisiae and has been named K1-like (K1L). Genomic homologs of K1L were identified in six non-Saccharomyces yeast species of the Saccharomycotina subphylum, predominantly in subtelomeric regions of the genome. When ectopically expressed in S. cerevisiae from cloned cDNAs, both K1L and its homologs can inhibit the growth of competing yeast species, confirming the discovery of a family of biologically active K1-like killer toxins. The sporadic distribution of these genes supports their acquisition by horizontal gene transfer followed by diversification. The phylogenetic relationship between K1L and its genomic homologs suggests a common ancestry and gene flow via dsRNAs and DNAs across taxonomic divisions. This appears to enable the acquisition of a diverse arsenal of killer toxins by different yeast species for potential use in niche competition.


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